ZAP-70 Enhances Migration of Malignant B Cells towards Lymphoid Organs in a Burkitt Lymphoma Xenograft Model

Abstract 2844

Introduction. ZAP-70 (ξ-associated protein) is a protein tyrosine kinase of the Syk/ZAP family that plays a crucial role in cellular activation in T and NK cells. High expression of ZAP-70 protein in malignant cells from Chronic Lymphocytic Leukemia (CLL) correlates with adverse clinical prognostic features, such as unmutated IgHV genes, short time to progression, and short survival. Moreover, ZAP-70 protein has been related to aggressive features of the CLL cells, such as enhanced B-cell receptor (BCR) signaling and higher migration capacity. To further investigate into the mechanisms by which ZAP-70 protein influences the clinical outcome of patients with CLL, we analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells. For this, Ramos and Raji (Burkitt) B-cell lines were stably transfected with a ZAP-70 expressing vector (pEGFP-N2ZAP-70). Raji transfectant showed constitutively phosphorylated ZAP-70 protein, whilst Ramos cells required stimulation with 5 μg/ml F(ab') 2 anti-IgM to get ZAP-70 activated. ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21 (own data, Blood 2011 pre-published). CCR7 ligands (chemokines CCL21 and CCL19) are mainly expressed in high endothelial venules and the T zones from secondary lymphoid organs. The aims of this study were firstly to evaluate in vivo the migratory/invasive capability of pEGFP-N2ZAP-70 transfected Raji and Ramos cell lines compared to pEGFP Raji and Ramos cell lines; and later, to compare the overall survival (OS) of mice injected with pEGFP-N2ZAP-70 transfected cells to those injected with only pEGFP transfected cells. Methods. For this, a total of 27 7- to 8-week old SCID (CB17Crl) mice were used. Mice were inoculated intravenously with 5×10⁶ cells of each cell line (6 mice with Raji-GFP, 5 mice with Raji-GFP-ZAP-70, 5 mice with Ramos-GFP and 10 mice with Ramos-GFP-ZAP-70). Mice were observed for the onset of hind legs paralysis, dyspnea, or evidence of tumor growth, once symptoms appeared, mice were euthanized and lymphoid and non-lymphoid organs were obtained for further analysis of the presence of GFP-positive cells by flow cytometry and immunohistochemistry. Results. Twenty-six out of twenty-seven injected mice were included in the analysis. The excluded mouse was found dead before it could be euthanized to obtain the organs. In the Raji xenograft model, 11/11 (100%) of mice had hind legs paralysis as the first symptom to appear. The median survival was 19 days for GFP-ZAP-70 and 16 days for GFP injected mice. There were no statistically significant differences between survival of GFP-ZAP-70 and GFP injected mice (OS was 66.7% [95% CI 38.4–100] vs 33.3% [95% CI 0–71.1], p=0.784, at 19 and 16 days, respectively). In the Ramos xenograft model, 6/15 (40%) of mice showed hind legs paralysis as the first symptom to appear, as well as evidence of abdominal tumor growth in 6/15 (40%), whereas in 3/15 (20%) the established event was dyspnea. The median survival in Ramos xenograft model was 40 days for GFP-ZAP-70 and 38 days for GFP injected mice. Again there were no statistically significant differences between survival of GFP-ZAP-70 and GFP Ramos injected mice (OS was 50% [95% CI 18.4–81.6] vs 40% [95% CI 0–83.8], p=0.180, at 40 and 38 days, respectively). By flow cytometry analysis of GFP cells we found that in the Raji xenograft model there were statistically significant differences between the migration of GFP-ZAP-70 and GFP injected cells towards bone marrow (21.5% vs 5.17, p=0.011), spleen (0.08% vs 0.01%, p=0.006) and thymus (0.00% vs 0.02%, p=0.037). The highest percentages of GFP positive cells were found in bone marrow samples (mean, 9.85%), whereas in spleen and thymus the percentages of GFP positive cells were all below 0, 1%. There was no statistically significant difference between the cellular migration in the Ramos xenograft model in any of the organs analyzed. Conclusion. In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein show enhanced ability to migrate towards and infiltrate lymphoid organs in a xenograft model, specially the bone marrow, although it does not translate into a worse survival of the animals. Further specific immunohistochemical assays to determine infiltrated areas by ZAP-70 expressing lymphocytes are in process.